Amanda Formosa

MicroRNAs, miR-154, miR-299-5p, miR-376a, miR-376c, miR-377, miR-381, miR-487b, miR-485-3p, miR-495 and miR-654-3p, mapped to the 14q32.31 locus, regulate proliferation, apoptosis, migration and invasion in metastatic prostate cancer cells

miRNAs act as oncogenes or tumor suppressors in a wide variety of human cancers, including prostate cancer (PCa). We found a severe and consistent downregulation of miRNAs, miR-154, miR-299-5p, miR-376a, miR-376c, miR-377, miR-381, miR-487b, miR-485-3p, miR-495 and miR-654-3p, mapped to the 14q32.31 region in metastatic cell lines as compared with normal prostatic epithelial cells (PrEC). In specimens of human prostate (28 normals, 99 primary tumors and 13 metastases), lower miRNA levels correlated significantly with a higher incidence of metastatic events and higher prostate specific antigen (PSA) levels, with similar trends observed for lymph node invasion and the Gleason score. We transiently transfected 10 members of the 14q32.31 cluster in normal prostatic epithelial cell lines and characterized their affect on malignant cell behaviors, including proliferation, apoptosis, migration and invasion. Finally, we identified FZD4, a gene important for epithelial-to-mesenchymal transition in (PCa), as a target of miR-377.

DNA methylation silences miR-132 in prostate cancer

Silencing of microRNAs (miRNAs) by promoter CpG island methylation may be an important mechanism in prostate carcinogenesis. To screen for epigenetically silenced miRNAs in prostate cancer (PCa), we treated prostate normal epithelial and carcinoma cells with 5-aza-2′-deoxycytidine (AZA) and subsequently examined expression changes of 650 miRNAs by megaplex stemloop reverse transcription–quantitative PCR. After applying a selection strategy, we analyzed the methylation status of CpG islands upstream to a subset of miRNAs by methylation-specific PCR. The CpG islands of miR-18b, miR-132, miR-34b/c, miR-148a, miR-450a and miR-542-3p showed methylation patterns congruent with their expression modulations in response to AZA. Methylation analysis of these CpG islands in a panel of 50 human prostate carcinoma specimens and 24 normal controls revealed miR-132 to be methylated in 42% of human cancer cases in a manner positively correlated to total Gleason score and tumor stage. Expression analysis of miR-132 in our tissue panel confirmed its downregulation in methylated tumors. Re-expression of miR-132 in PC3 cells induced cell detachment followed by cell death (anoikis). Two pro-survival proteins—heparin-binding epidermal growth factor and TALIN2—were confirmed as direct targets of miR-132. The results of this study point to miR-132 as a methylation-silenced miRNA with an antimetastatic role in PCa controlling cellular adhesion.

miR-143 regulates hexokinase 2 expression in cancer cells

Tumor cells activate pathways that facilitate and stimulate glycolysis even in the presence of adequate levels of oxygen in order to satisfy their continuous need of molecules, such as nucleotides, ATP and fatty acids, necessary to support their rapid proliferation. Accordingly, a variety of human tumors are characterized by elevated expression levels of the hexokinase 2 isoform (HK2). Although different molecular mechanisms, including genetic and epigenetic mechanisms, have been suggested to account for the altered expression of HK2 in tumors, the potential role of microRNAs (miRNAs) in the regulation of HK2 expression has not been evaluated. Here, we report that miR-143 inhibits HK2 expression via a conserved miR-143 recognition motif located in the 3′-untranslated region (3′UTR) of HK2 mRNA. We demonstrate that miR143 inhibits HK2 expression both in primary keratinocytes and in head and neck squamous cell carcinoma (HNSCC)-derived cell lines. Importantly, we found that miR-143 inversely correlates with HK2 expression in HNSCC-derived cell lines and in primary tumors. We also report that the miRNA-dependent regulation of hexokinase expression is not limited to HK2 as miR-138 targets HK1 via a specific recognition motif located in its 3′UTR. All these data unveil a new miRNA-dependent mechanism of regulation of hexokinase expression potentially important in the regulation of glucose metabolism of cancer cells.

Epigenetically silenced miR-34b/c as a novel faecal-based screening marker for colorectal cancer

Background:MicroRNAs are tiny non-coding small endogenous RNAs that regulate gene expression by translational repression, mRNA cleavage and mRNA inhibition. The aim of this study was to investigate the hypermethylation of miR-34b/c and miR-148a in colorectal cancer, and correlate this data to clinicopathological features. We also aimed to evaluate the hypermethylation of miR-34b/c in faeces specimens as a novel non-invasive faecal-DNA-based screening marker.Methods:The 5-aza-2′-deoxycytidine treatment and methylation-specific PCR were carried out to detect the hypermethylation of miR-34b/c and miR-148a.Results:The miR-34b/c hypermethylation was found in 97.5% (79 out of 82) of primary colorectal tumours, P=0.0110. In 75% (21 out of 28) of faecal specimens we found a hypermethylation of miR-34b/c while only in 16% (2 out of 12) of high-grade dysplasia. In addition, miR-148a was found to be hypermethylated in 65% (51 out of 78) of colorectal tumour tissues with no significant correlation to clinicopathological features. However, a trend with female gender and advanced age was found, P=0.083. We also observed a trend to lower survival rate in patients with miR-148a hypermethylation with 10-year survival probability: 48 vs 65%, P=0.561.Conclusions:These findings show that aberrant hypermethylation of miR-34b/c could be an ideal class of early screening marker, whereas miR-148a could serve as a disease progression follow-up marker.

Novel pharmacokinetic behavior of intravenous busulfan in children with thalassemia undergoing hematopoietic stem cell transplantation: a prospective evaluation of pharmacokinetic and pharmacodynamic profile with therapeutic drug monitoring

We prospectively studied the pharmacokinetics (PK) and clinical outcomes of intravenous busulfan (Bu) in 71 children with preexisting liver damage who underwent hematopoietic stem cell transplantation for thalassemia. Intravenous Bu was administered every 6 hours as part of a conditioning regimen with PK-based dose adjustment to target a conservative area under the concentration-versus-time curve (AUC) range (900-1350 microMol*min). The first-dose Bu clearance (CL) was significantly higher than the subsequent daily CL that remained unchanged in the ensuing days. One-third of patients required dose escalation based on dose 1 AUC, whereas dose reduction was needed in the subsequent days. At doses 5, 9, and 13, 78%, 81%, and 87% of patients, respectively, achieved the target range of AUC. A population PK analysis confirmed that the first-dose CL was 20% higher and that body weight was the most important covariate to explain PK variability. Patients with variant GSTA1*B had a 10% lower Bu CL than wild-type. These results suggest that the disease-specific behavior of intravenous Bu PK should be considered for PK-guided dose adjustment in patients with thalassemia, and the use of a conservative AUC range resulted in low toxicity, good engraftment, and good survival rate.

A simplified, non-invasive fecal-based DNA integrity assay and iFOBT for colorectal cancer detection.

PurposeNeoplasia cells exfoliated from colorectal epithelium have dysfunctional apoptotic mechanisms, thus it is possible to identify high-molecular weight DNA fragments in feces. This prospective single-center study was performed to evaluate the sensitivity and specificity of fecal-based DNA integrity versus immunological fecal occult blood test (iFOBT) and calprotectin for colorectal cancer (CRC) and adenoma detection.MethodsFeces were collected from 204 subjects and DNA integrity was quantified by quantitative-denaturing high performance liquid chromatography (QdHPLC). Calprotectin and iFOBT were assessed using commercial kits. The diagnostic performance was calculated by receiver operating characteristic (ROC) curves analysis.ResultsA total of 192 fecal specimens were analyzed and 12 samples were excluded due to DNA degradation. We found long DNA (L-DNA) occurrence in feces with a sensitivity of 86% (n = 24/28) and a specificity of 81% for CRC detection. To minimize false-positive cases of the developed test, area under the curve of ROC was evaluated such that the specificity was increased to 92% with decreased sensitivity to 79%, p = 0.0001 for CRC detection. iFOBT was positive in 51% (n = 14/27) while calprotectin was positive in 75% (n = 18/27). The combination of iFOBT and L-DNA identified a greater number of CRC cases with a sensitivity of 89% and a specificity of 95%, p < 0.001. The combination also improved the sensitivity of polyps, particularly high-grade dysplasia and advanced adenoma (33%, p = 0.0015) as opposed to a single evaluation assay (17–21%).ConclusionsThis study illustrates the usefulness of fecal DNA integrity assay by QdHPLC as a non-invasive, easy-to-perform, and reproducible method with a high level of sensitivity in detecting individuals with colorectal neoplasia. Combination of iFOBT and L-DNA improves the sensitivity for CRC and adenoma detection.

Plasmin system of Alzheimer’s disease patients: CSF analysis

Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder characterized by the extracellular deposit of Amyloid beta (Aβ), mainly of the Amyloid beta1–42 (Aβ1–42) peptide in the hippocampus and neocortex leading to progressive cognitive decline and dementia. The possible imbalance between the Aβ production/degradation process was suggested to contribute to the pathogenesis of AD. Among others, the serine protease plasmin has shown to be involved in Aβ1–42 clearance, a hypothesis strengthened by neuropathological studies on AD brains. To explore whether there is a change in plasmin system in CSF of AD patients, we analyzed CSF samples from AD and age-matched controls, looking at plasminogen, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) protein levels and t-PA and urokinase plasminogen activator (u-PA) enzymatic activities. We also measured Aβ1–42, total-tau and phospho-tau 181 CSF levels and sought for a possible relationship between them and plasmin system values. Our findings showed that t-PA, plasminogen and PAI-1 levels, as t-PA enzymatic activity, remained unchanged in AD with respect to controls; u-PA activity was not detected. We conclude that CSF analysis of plasminogen system does not reflect changes observed post-mortem. Unfortunately, the CSF detection of plasmin system could not be a useful biomarker for either AD diagnosis or disease progression. However, these findings do not exclude the possible involvement of the plasmin system in AD.

Salivary miRNAome profiling uncovers epithelial and proliferative miRNAs with differential expression across dentition stages

Saliva’s ability to mirror the internal physiological environment of an organism coupled with its facile accessibility makes it an attractive diagnostic medium. The finding of microRNAs (miRNAs) in saliva has expanded the field of biomarker discovery since these tiny non-coding RNAs affect various physiological processes and diseases. Few reports have linked miRNAs to tooth development and eruption, with none having studied this in humans. As a first initiative to describe miRNAs in saliva whose modulations may reflect developing and erupting teeth, we quantified the levels of 730 miRNAs in the saliva of children of varying dentition stages: edentulous (newborns), deciduous and permanent by megaplex stemloop reverse-transcription quantitative PCR. The three groups expressed 193, 181 and 192 miRNAs, respectively, where 125 miRNAs had consistent expression. The remaining miRNAs had inter-group variations from 5 to hundreds of fold, where most had either an increasing or decreasing trend in going from edentulous to deciduous to permanent. A literature survey of epithelial miRNAs found most were present in saliva. Moreover, most miRNAs with expression differences between groups had previously documented functions in proliferation, cell cycle, apoptosis and other cellular behaviors key to the dynamics of tooth morphogenesis. Lastly, miRNAs of the same family, such as the let-7 and miR-200 families, or transcribed from the same hairpin, had similar expression patterns. The results presented here should serve as a salivary miRNA dictionary for future studies in tooth development as well as in childhood diseases associated with modulations in saliva composition.

Quantitative denaturing high performance liquid chromatography (Q-dHPLC) detection of APC long DNA in faeces from patients with colorectal cancer.

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer-related deaths. However, prevention is possible by early detection. In the present work, we have demonstrated and validated a novel quantitative method based on a DNA integrity assay and mutation in faeces of CRC patients using denaturing high performance liquid chromatography (dHPLC). Methods: Faecal DNA (fDNA) was isolated from 28 CRC, 96 healthy and 61 patients with adenomas. Adenomatosis polyposis coli (APC)-Long-DNA and its mutations were analysed using dHPLC and the Sanger sequencing method. The diagnostic performance was assessed using receiver operating characteristic curve analysis. Results: We detected APC-Long-DNA in 21/28 CRC subjects with a sensitivity of 75% and specificity of 91.7%. A cut-off ratio of 0.2317 was used for APC/β-actin. The Q-dHPLC detection limit was 0.02 ng/injection. The average initial fDNA presence based on a single gene of β-actin was 26.12±13.39 ng/mL for healthy, and 49.61±46.28 ng/mL for CRC subjects, with a sensitivity of 71.4% and a specificity of 84.4% at a cut-off value >29 ng/mL. We also detected a novel mutation at codon 1576 Lys/Glu using dHPLC. Conclusions: This study highlights a novel application of Q-dHPLC in the DNA integrity assay, which demonstrates high performance, good reproducibility, and low cost for the CRC detection using faeces. Further studies in a larger population are needed to confirm these results. Clin Chem Lab Med 2010;48:1303–11.